Carboxylic acid-type lipid

ABSTRACT

As a negatively charged lipid that can stably add negative charges to the surface of vesicles without side effects, the carboxyl acid-type lipid of the following general formula [1] 
                 
 
     [wherein R 1 , R 2  and R 3  represent substituents of which one is represented by the following general formula [X]:  
                 
 
     (wherein M is a hydrogen atom or monovalent cation, and m is an integer of 1 to 5 that represents the methylene chain length), and the other two are chained hydrocarbon groups; A 1 , A 2  and A 3  are the same or different substituents selected from the group consisting of C(O)O, CONH or NHCO; and n is an integer of 1 to 3 that represents the methylene chain length) is provided.

TECHNICAL FIELD

[0001] The invention of the present application relates to a carboxylicacid-type lipid that may be used as membrane lipids of vesicles. Inparticular, the invention of the present application relates to anegatively charged carboxylic acid-type lipid that may be used as thenegatively charged component of molecular assemblies and molecularassembly membranes of vesicles, which enables the adjustment of surfacecharge density and surface hydration of the assembly, controlling of thenumber of layers of the multi-lamellar vesicle, and inhibition ofinter-vesicle aggregation, depending on their structure and composition.

BACKGROUND ART

[0002] Vesicles encapsulating useful substances in the internal aqueousphase and their dispersions are an important technology in variousfields such as pharmaceuticals, perfumes, cosmetics and food stuffs.Examples of widely used lipids that constitute the membrane of thevesicle include mixed lipids prepared by mixing negativ ly chargedphospholipids such as diacylphosphatidyl glycerol, diacylphosphatidylinositol, diacylphosphatidyl serine with diacylphosphatidyl choline or amixture of diacylphosphatidyl choline and cholesterol in an arbitraryproportion.

[0003] However, diacylphosphatidyl glycerol, diacylphosphatidylinositol, diacylphosphatidyl serine and diacylphosphatidyl ethanolaminethat are currently introduced as negatively charged components inbiologically applicable vesicle preparations are reported to induceaggregation of platelets in the body because they are physiologicallyactive. It has been elucidated that administration of the vesiclepreparations also causes severe side effects such as thrombocytopeniaand dysfunction of white blood cells.

[0004] While long chain fatty acids have been used for a simplifiedmethod of introducing negative charges on the surface of the vesiclewithout using any negatively charged phospholipids, single chain fattyacids cannot be stably introduced into the molecular assembly. Instead,there is a problem that a part of the fatty acid leaks into the aqueousphase, or is extracted by lipoproteins and albumin in the blood.

[0005] Taking into account the above situations, the object of theinvention of the present application is to solve the problems of theprior art, and to provide a negatively charged lipid that can stably addnegative charges co the surface of a vesicle without any side effects.

DISCLOSURE OF INVENTION

[0006] In light of the above-mentioned circumstance, the inventors ofthe present invention achieved, as a result of intensive studies, thesynthesis of carboxylic acid-type double chain lipids containing nophosphate groups using a compound that contains at least threefunctional groups as a spacer. The inventors also found that thecarboxylic acid-type double chain lipid is stably fixed in phospholipidbilayer membranes, and prevents platelets from aggregating in the bloodwhen the vesicles to which the carboxylic acid-type lipid is introducedare used as negatively charged components, thereby completing theinvention of the present application.

[0007] Hence, as a means to solve the above-described problems, theinvention of the present application firstly provides a carboxylicacid-type lipid represented by the following general formula (1):

[0008] [wherein R₁, R₂ and R₃ represent substituents of which one isrepresented by the following general formula [X]:

[0009] (wherein M is a hydrogen atom or monovalent cation, and m is aninteger of 1 to 5 that repres nts th methylene chain length), and theother two are chained hydrocarbon groups; A₁, A₂ and A₃ are the same ordifferent substituents selected from the group consisting of C(O)O, CONHor NHCO; and n is an integer of 1 to 3 that represents the methylenechain length].

[0010] Further, secondly, the invention of the present applicationprovides a carboxylic acid-type lipid represented by the followinggeneral formula [2]:

[0011] [wherein R₁, R₂ and R₃ represent substituents of which one isrepresented by the following general formula EX):

[0012] (wherein M is a hydrogen atom or monovalent cation, and m is aninteger of 1 to 5 that represents the methylene chain length), and theother two are chained hydrocarbon groups; A₁, A₂ and A₃ are the same ordifferent substituents selected from the group consisting of OC(O) andO; and n and n, are integers of 1 to 3 that represent the methylenechain length).

[0013] The invention of the present application thirdly provides acarboxylic acid-type lipid represented by the general formula [3]:

[0014] [wherein M is a hydrogen atom or monovalent cation; m is aninteger of 1 to 5 that represents the methylene chain length; R₁ and R₂are chained hydrocarbon groups; A₁ and A₂ are the same or differentsubstituents selected from the group consisting of C(O)O and CONH; and nis an integer of 1 to 3 that represents the methylene chain length].

[0015] Fourthly, the invention of the present application provides acarboxylic acid-type lipid represented by the following general formula[4];

[0016] (wherein R₁, R₂ and R₃ represent substituents of which one isrepresented by the following general formula [x]:

[0017] (wherein M is a hydrogen atom or monovalent cation, and m is aninteger of 1 to 5 that represents the methylene chain length), and theother two are chained hydrocarbon groups; A₁, A₂ and A3 are the same ordifferent substituents selected from the group consisting of C(O)O andCONH; and R₄ is selected from the group consisting of a hydrogen atom,methyl group and acetylene group].

[0018] The invention of the present application fifthly provides acarboxylic acid-type lipid represented by the following general formula[5]:

[0019] [wherein R₁, R₂ and R₃ are substituents of which one isrepresented by the following general formula [X]:

[0020] (wherein M is a hydrogen atom or monovalent cation, and m is aninteger of 1 to 5 that represents the methylene chain length), and theother two are chained hydrocarbon groups; A₁, A₂ and A₃ are the same ordifferent substituents selected from the group consisting of OC(O). O,NH, CONH and NHCO; and D represents a saccharide].

[0021] Further, sixthly, the invention of the present applicationprovides a carboxylic acid-type lipid represented by the followingformula (6a] or [6b];

[0022] [wherein either one of R₁ or R₂ is a hydrogen atom and the otheris OR′″; either one of R₃ and R₄ is a hydrogen atom and the other isOR′″; R, R′, R″, R′″ and R″″ are substituents of which one isrepresented by the following general formula [Y];

[0023] (wherein M is a hydrogen atom or monovalent cation, A is CH₂ orCO, and m is an integer of 1 to 5 that represents the methylene chainlength), at least two are chained hydrocarbon groups, and the others arehydrogen atoms; and n is an integer of 1 to 3 that represents the degreeof polymerization].

[0024] Furthermore, the invention of the present application providesseventhly, a carboxylic acid-type lipid represented by the followinggeneral formula [7]:

[0025] (wherein R₁ is an aliphatic hydrocarbon group, F is a monodendronconstituting unit, R₂ is a linker, M is a hydrogen atom or monovalentcation, m is an integer of 1 to 5 that represents the methylene chainlength, p is an integer of 2 and q is an integer of 1 to 4 thatrepresents the number of repeating units in the dendron); and eigthly,the carboxylic acid-type lipid of th seventh invention, wherein themonodendron constituting unit is one or more amino acid,

[0026] Ninthly, the invention of the present application provides acarboxylic acid-type lipid represented by the following general formula[8]

[0027] (wherein M is a hydrogen atom or monovalent cation, m is aninteger of 1 to 5 that represents the methylene chain length, and n andn′ are integers of 13 to 21 that represent the chain lengths ofmethylene).

BEST MODE FOR CARRYING OUT THE INVENTION

[0028] The invention of present application provides the carboxylicacid-type lipids represented by the general formula [1] to [8].

[0029] In the carboxylic acid-type lipid represented by the generalformula [1], the bonding site of R₁, R₂ and R₃ are preferablytrifunctional amino acids. Specific examples of the amino acids includelysine, asparagine, glutamine, aspartic acid, glutamic acid, serine,threonine and tyrosine. Trifunctional amino acids having one reactivefunctional group and two equivalent functional groups (for example:aspartic acid and glutamic acid, which contain one terminal amino groupand two terminal carboxylic groups; or lysine, glutamine and asparagine,which contain one terminal carboxylic group and two terminal aminogroups) are preferable and glutamic acid and aspartic acid areparticularly preferable. The amino acids may be homocysteine andglutathione.

[0030] For the carboxylic acid-type lipid represented by the generalformula [2] of the invention of the present application, the bondingsite of R₁, R₂ and R₃ is preferably glycerol. Further, for thecarboxylic acid-type lipid represented by the general formula [3] of theinvention of the present application, maleic acid is particularlypreferable an the bonding site of R₁ and R₂. For the carboxylicacid-type lipid represented by the general formula [4], citric acid ispreferably used as the bonding site of R₁, R₂ and R₃.

[0031] In the carboxylic acid-type lipid represented by the generalformula [5] of the invention of the present application, the bondingsite D is a saccharide. Further, in the carboxylic acid-type lipidrepresented by the general formula [6], the bonding sites of R₁ to R₄are also a saccharide. Any saccharides may be used as long as they canbe made hydrophobic by covalent bonds, and may be selected from variousnatural and synthetic saccharides. Examples of such saccharides aresaccharides obtained by the α- or β-bonds of at least two monosaccharideunits such as glucose, fructose, xylose, galactose, mannose andglucosamine, particularly maltose, cellobioge, lactose, xylobiose,isomaltose, gentiobiose, melibiose, boranteobiose, methinol,primeverose, bicyanose, nigerose, laminaribiose, turanose, kojibiose,sophorose, sucrose, trehalose, chitobiose, hyalurobiouronic acid,chondrosin, cellobiouronic acid, malto-oligosaccharide,laminario-oligosaccharide, cello-oligosaccharide,isomalto-oligosaccharide, gentio-oligosaccharide,nigero-oligosaccharide, lacto-oligosaccharide, meli-oligosaccharide andinulo-oligosaccharide; polysaccharide such as starch, pullulan,cellulose, muco-polysaccharide (hyaluronic acid, chondroitin,chondroitin sulfate, delmantan sulfate, ketaran sulfate, heparin),chitin and chitosan; and further, decomposition products ofpolysaccharides, cell and complex saccharides derived from bacteria.Among these, monosaccharides are preferable.

[0032] As the binding site (F) of the carboxylic acid-type lipidrepresented by the general formula [7] of the invention of the presentapplication monodendron may be used. Such monodendron contains aminogroups, carboxylic groups and hydroxyl groups at the branch terminus,and amino groups, carboxylic groups and hydroxyl groups as the core.Monodendrons with 1 to 5 branch generations are preferable. While F ispreferably an amino acid such as lysine and glutamic acid wh n themonodendron is used as a biocompatible material, it is not restricted tothem.

[0033] In the invention of the present application, among thesubstituents R₁ to R₄, one is represented by the above-described generalformula [X] or [Y], and the rest are linear hydrocarbon groups. In thegeneral formula [x] and [Y], M may be a hydrogen atom or a monovalentcation such as (although not limited to) Na⁻, K⁻, Li⁻ and NH₃ ⁻.

[0034] Furthermore, when the substituents of RX to R₄ are linearhydrocarbon groups, they may be hydrophobic groups introduced into thefunctional groups of amino acids, glycerol, maleic acid, citric acid,saccharides, cysteine, and monodendron by covalent bonding: straight orbranched linear hydrocarbon groups with 1 to 30 carbons are preferableamong them. Further, these groups may contain substituents such ascarboxylic groups, hydroxyl groups and amino groups. When the linearhydrocarbon group contains unsaturated bonds, its number may preferablybe 1 to 4.

[0035] Examples of the starting material for the above linearhydrocarbon group include saturated linear fatty acids containingcarboxylic groups such as: caprylic acid, undecanoic acid, lauric acid,tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid,margarine acid, stearic acid, nonadecanoic acid, arachidic acid, behenicacid, lignoceric acid, cerotic acid, montanic acid and melissic acid;and unsaturated linear fatty acids such as obtusilic acid, lindericacid, thujic acid, fisetleic acid, palmitoleic acid petroceric acid,erucic acid, oleic acid, eladic acid, vaccenic acid, linolic acid,nervonic acid, linoelaidic acid, linolenic acid, γ-linolenic acid,bishomo-γ-linolenic acid and arachidonic acid. These may containbranched chains. Examples of branched fatty acid include iso-acids suchas iso-lauric acid, iso-myristic acid, iso-palmitic acid, iso-stearicacid and iso-arachidic acid; and antiiso-acids such as 9-methylundecanoic acid, 10-methyl dodecanoic acid, 11-methyl tridecanoic acid,12-methyl tetradecanoic acid, 13-methyl pentadecanoic acid, 14-methylhexadecanoic acid, 15-methyl heptadecanoic acid and 16-methyloctadecanoic acid. These acids may also be anhydrides and chlorides ofthese acids.

[0036] Further, examples of the starting materials of the linearhydrocarbon groups in the carboxylic acid-type lipid of the presentinvention include linear primary saturated alcohols containing hydroxylgroups such as decanol, lauryl alcohol, cetyl alcohol, stearyl alcohol,eicosanol, docosanol, tetracosanol, hexacosanol, octacosanol,nonacosanol and myristyl alcohol. Further examples include linearunsaturated alcohols, branched primary saturated alcohols, branchedprimary unsaturated alcohols, secondary saturated alcohols and secondaryunsaturated alcohols examples of which are dodecenol, ficeterialalcohol, zomaryl alcohol, oleyl alcohol, gadoleil alcohol, 11-eicosenol,11-docosenol, 13-docosenol, 15-tetracosenol, cathadonyl alcohol andlinolenyl alcohol. Further examples include dialkyl glycerols comprisingthe primary saturated alcohols or primary unsaturated alcohols bonded to1,3-position or 1,2-position of glycerin.

[0037] Further, examples of linear primary amines containing an aminogroup include dodecyl amine, tridecyl amine, tetradecyl amine,pentadecyl amine, hexadecyl amine, heptadecyl amine, octadecyl amine,docosyl amine and oleyl amine. These amines may contain branched chains.

[0038] In the carboxyl acid-type lipid of the present invention, thehydrophilic groups may be those that can be introduced into thefunctional groups of amino acids, glycerol, maleic acid, saccharides,cysteine and monodendron by covalent bond with or without theinterposition of a spacer of an arbitrary length such as methylenegroups or oxyethylene groups. The length of the methylene group,oxymethylene group, or the like may be approximately 0 to 1000,preferably, 1 to 5. Preferable examples of such hydrophilic groupsinclude malonic acid, succinic acid, glutaric acid, adipic acid, pimelicacid and its anhydride, glycolic acid, 3-hydroxypropionic acid,4-hydroxybutyric acid, 5-hydroxyvaleric acid. 6-hydroxycaproic acid,bromoacetic acid, 3-bromopropionic acid, 4-bromobutyric acid,5-bromovaleric acid and 6-bromocaproic acid (ethyleneglycol oligomerswith both t rminus carboxylated, and ethyleneglycol oligomerscarboxylated at one terminal).

[0039] While the bonding positions A₁, A₂ and A3 to be used in thecarboxylic acid-type lipid of the present invention may independently beselected from, an ester, ether, amide and imide, they are not restrictedto these. A₁, A₂ and A₃ may be the same or different with each other.

[0040] Embodiments of the present invention will be described in furtherdetail by means of examples with reference to the attached drawings.However, it is needless to say that the invention is not restricted tothe examples below, and various aspects are possible with respect to thedetails.

EXAMPLE Example 1 The First Invention

[0041] Glutamic acid (2.96 g, 20 mmol) p-toluene sulfonic acid (4.56 g,24 mmol) and hexadecyl alcohol (10.65 g, 44 mmol) were dissolved inbenzene (150 mL), and the solvent was refluxed at 100° C. for 14 hourswhile dehydrating the reaction system.

[0042] After removing the solvent under a reduced pressure, the reactionproduct was dissolved in chloroform, and the solution was washed threetimes with a saturated aqueous sodium hydrogencarbonate solution andthree times with water, respectively.

[0043] The chloroform layer was dehydrated using sodium sulfate,filtrated, aft r which the solvent was removed under reduced pressure.The residue was dissolved in methanol (400 mL) at 60° C. andrecrystallized at 4° C., after which the precipitate was filtrated anddried, where by a glutamic acid derivative [A] was obtained as a whitesolid (9.5 g, yield 80%). Compound [A] in which tetradecyl alcohol oroctadecyl alcohol was bonded to glutamic acid was also synthesized bythe same method

[0044] The identification results are shown in Table 1. TABLE 1 Thinlayer chromatography (silica gel plate, chloroform/methanol 4/1(volume/volume)): Rf: 0.79 (monospot). Infrared absorption spectra(cm⁻¹): 3385[V_(N—H) (NH₂)]; 1783[V_(C—O)(ester)]. ¹H—NMR spectra[CDCl₃,500MHz, δ (ppm)]: 0.88(t, 6H, —CH₃); 1.26(s, 52H, —CH₂—CH₂—); 1.62(m,4H, CO—O—C—CH₂); 1.85, 2.08(m, 2H, glu β-CH₂); 2.46(m, 2H, glu γ-CH₂);3.46(m, 1H, glu α-CH); 4.08, 4.12 (tt, 4H, —CO—O—CH₂—).

[0045] The resulting compound [A] (13.49 g, 2.5 mmol) was dissolved in amixed solution of chloroform and tetrahydrofuran (mixing ratio 1:1(volume/volume)), and the solution was stirred for 5 hours after addingsuccinic anhydride (0.38 g, 3.8 mmol). After removing the solvent undera reduced pressure, the reaction product was recrystallized from a mixedsolvent of ethanol and acetone (mixing ratio 1:5 (volume/volume)) at 4°C., after which the precipitate was filtrated and dried; a negativelycharged lipid [B] having a glutamic acid structure was obtained as awhite solid (1.5 g, yield 86%).

[0046] The identification results are shown in Table 2. TABLE 2 Thinlayer chromatography (silica gel plate, chloroform/methanol (4/1)(volume/volume)): Rf: 0.65 (monospot). Infrared absorption spectra(cm⁻¹) 3314[V_(N—H) (amide)]; 1734[V_(C—O)(ester)]. ¹H—NMRspectra[CDCl₃, 500MHz, δ (ppm)]: 0.88(t, 6H, —CH₃); 1.26(s, 52H,—CH₂—CH₂—); 1.63(m, 4H, —CO—O—C—CH₂); 2.04, 2.21(m, 2H, glu β-CH₂);2.40(m, 2H, —CH₂—CO—NH—); 2.58(m, 2H, glu γ-CH₂); 2.72(m,—CH₂—C—CO—NH—); 4.06, 4.14(tt, 4H, —CO—O—CH₂—); 4.60(m, 1H, glu α-CH);6.55(m, 1H, —CO—NH—). MS(FAB); calculated value for C₃₇H₇₃O₄N: 696.0;observed value: 696.5(M⁻H)⁻.

Example 2 The Second Invention

[0047] Glycerol (1.50 g, 32.6 mmol) and palmitic chloride (8.96 g, 32.6mmol) were dissolved in dry chloroform, and the solution was stirred for6 hours at room temperature under the presence of dimethylaminopyridine(3.98 g, 32.6 mmol). The reaction solution was washed with sodiumchloride solution, saturated aqueous sodium hydrogencarbonate solutionand saturated sodium chloride solution, after which the chloroform layerwas recovered and concentrated under reduced pressure, followed byrecrystallization from hexane. The crystal was filtrated, dissolved inchloroform, and further filtrated through a silica gel column (5×15 cm),whereby a glycerol derivative with palmitoyl groups at the 1- and3-positions were obtained (5.56 g, yield 60%).

[0048] The identification results are shown in Table 3. TABLE 3 Thinlayer chromatography (silica gel plate, chloroform): Rf 0.40,(monospot). Infrared absorption spectra (cm⁻¹): 3420[V_(O—H) (hydroxylgroup)]; 1748[V_(C—O)(ester)]. ¹H—NMR spectra[CDCl₃, 500MHz, δ (ppm)]:0.88(t, 6H, —CH₃); 1.26(s, 56H, —CH₂—CH₂—); 1.63(t, 4H, —CO—O— C—CH₂);3.1(m, 1H, glycerol CH); 3.40(m, 4H, glycerol CH₂).

[0049] The glycerol derivative [C] (4 g, 7.03 mmol) and adipic anhydride(1.03 g, 7.04 mmol) were dissolved in dry THF, and the solution wasrefluxed for 5 hours in the presence of a catalytic amount of DMAP (42mg, 0.35 mmol). After removing the solvent under reduced pressure, theresidue was dissolved in dichloromethane, and the solution was filteredto remove insoluble impurities followed by removing the solvent againunder reduced pressure. The residue was dissolved in chloroform, and thesolution was passed through a silica gel column to elute any unreactedcompound [C]. After confirming the completion of elution, the solventwas substituted by ethyl acetate, whereby compound [D] having palmitoylchains at the 1- and 3-positions, and a carboxylic group at the2-position was obtained at a yield of 80%.

[0050] The identification results for the glycerol derivative [D] areshown in Table 4. TABLE 4 Thin layer chromatography (silica gel plate,chloroform): Rf; 0.05 (monospot). Infrared absorption spectra (cm⁻¹):3321-2690[V_(O—H) (carboxylic acid); 1748[V_(C—O)(ester)]; 1695[V_(C—O)(carboxylic acid)]. ¹H—NMR spectra[CDCl₃, 500MHz, δ (ppm)]; 0.88(t, 6H,—CM₃) 1.26(s, 56H, —CH₂—CH₂—); 1.63(t, 4H, —CO—O—C— CH₂); 2.66(m, 8H,—CH₂ adipic acid); 3.33(m, 1H, glycerol —CH); 3.40(m, 4H, -glycerolCH₂).

Example 3 The Third Invention

[0051] Maleic acid (2.32 g, 20 mmol), p-toluene sulfonic acid (4.56 g,24 mmol) and hexadecyl alcohol (10.56 g, 44 mmol) were dissolved inbenzene (150 mL), and the solvent was refluxed at 100° C. for 6 hourswhile removing water generated by the reaction. The reaction solutionwas washed three times with saturated aqueous sodium hydrogencarbonatesolution and three times with water. After removing the solvent underreduced pressure, the product was recrystallized from methanol (400 mL)at 4° C., and the crystal was filtrated and dried, whereby the maleicacid derivative [E] with the alkyl groups esterified to the carboxylgroups was obtained as a white solid (8.46 g, yield 75%). Production ofthe desired compound was confirmed by the appearance of C═O stretchvibration (1740 cm⁻¹) attributed to the ester bond in the infraredabsorption spectrum.

[0052] The resulting maleic acid derivative [E] (5.65 g, 10 mmol) and3-mercaptopropionic acid (1.27 g, 12 mmol) were stirred at 25° C. for 24hours. The product was recrystallized from methanol (200 mL) at 4° C.,filtrated and dried, whereby a carboxylic acid double chain lipid (F)with a maleic acid structure was obtained as a white solid (4.68 g,yield 70%).

Example 4 The Fourth Invention

[0053] Citric acid (2.00 g, 10.5 mmol) was dissolved in 10 mL of aceticanhydride, and the solution was stirred at room temperature for 4 hoursin the presence of {fraction (1/20)} equivalence of DMAP. A citric acidderivative with its hydroxyl groups protected by acetyl group wasobtained by removing acetic anhydride under reduced pressure. Tracequantity of acetic acid odor disappeared by freeze drying from benzene.A peak attributing to an ester bond was confirmed in the IR spectrum at1578 cm^(−1 [v) _(C═O) (ester)] The resulting hydroxyl group-protectedcitric acid was dissolved and dispersed in 100 mL of benzene, andp-toluene sulfonic acid (198 mg, 1.05 mmol) and hexadecyl alcohol (2.54g, 208 mmol) were added to the solution. The mixture was reacted for 2hours under refluxing while dehydrating, using a Dean-Stark apparatus.After confirming the disappearance of the starting alcohol from TLC, thesolvent was removed under reduced pressure. The residue was dissolved inchloroform, and the solution was washed twice with 1M aqueous citricacid solution and twice with saturated sodium chloride solution. Afterdrying the solution with anhydrous sodium sulfate, chloroform wasremoved under reduced pressure. The residue was dissolved in hotmethanol and recrystallized, whereby compound [G] with palmitoyl chainsat the 1- and 3-positions, and carboxylic and acetyl groups at the2-position was obtained (5.26 g, 74%).

[0054] The identification results for the citric acid derivative [G] areshown in Table 5. TABLE 5 Thin layer chromatography (silica gel plate,chloroform): Rf: 0.05 (monospot). Infrared absorption spectra (cm⁻¹):3321-2690[V_(O—H) (carboxylic acid)]; 1748[V_(C—O)(ester)]; 1695[V_(C—O)(carboxylic acid)]. MS (FAB): calculated value based on C₄₀H₇₄O₆: 683.0;observed value: 682.5 (M⁻H)⁻. ¹H—NMR spectra(CDCl₃, 500MHz, δ (ppm)):0.92(t, 6H, —CH₃), 1.26(s, 56H, —CH₂—CH₂—); 1.63(t, 4H, —CO—O— CH₂);1.99(s, 3H, —COCH₃); 2.80(m, 4H, citric acid —CH₂); 3.97(t, 4H,CO—O—CH₂).

Example 5 The Sixth Invention

[0055] Glucose (0.9 g, 5.0 mmol) was dissolved in dehydratedN,N′-dimethyl formamide (20 mL) and pyridine (2 mL), and stearic acidchloride (3.64 g, 12.0 mmol) was added dropwise to the solution. Afterstirring the reaction mixture for 3 hours while maintaining atemperature of 80° C., methanol (50 mL) was added to the reactionsolution. The precipitate was recovered by filtration, washed with waterand dried, whereby the saccharide derivative [H] with alkyl groupsesterified to the hydroxyl groups was obtained as a white solid (2.67 g,yield 75%). Production of the desired compound was confirmed by theappearance of the C═O stretch vibration (1740 cm⁻¹) attributing to theester bond in the infrared spectrum. The number of alkyl groups perglucose was calculated to be 2.1 from the nuclear magnetic resonancespectrum.

[0056] The resulting saccharide derivative [H] (2.14 g, 3.0 mmol) andsuccinic anhydride (0.36 g, 3.6 mmol) were dissolved in dehydratedN,N′-dimethyl formamide (20 mL), and the solution was stirred for 6hours at 25° C. After removing the solvent under reduced pressure, theproduct was recrystallized from methanol (200 mL) at 4° C., filtratedand dried, and the carboxylic acid double chain lipid of [I) with asaccharide structure was obtained as a white solid (1.59 g, yield 70%).The number of uccinic acid bonded thereto per glucose was calculated tobe 1.0 from the nuclear magnetic resonance spectrum.

Example 6 The Seventh Invention

[0057] L-glutamic acid (1.47 go 10 mmol) and t-butoxycarbonyl anhydride(2.62 g, 12 mmol) were dissolve in a mixed solvent of dioxane (20 mL),water (10 ML) and 1N—NaOH (10 mL), and the solution was stirred at 25°C. for 6 hours. The solution was concentrated to 10 mL under reducedpressure, and 5% aqueous potassium hydrogen sulfate was added until thepH of the solution became 2.4, after which the solution was washed threetimes each with ethyl acetate, and water. After dehydrating the ethylacetate layer with sodium sulfate, the solvent was removed under reducedpressure. The residue was recrystallized from hexane at 4° C., and- thecrystal was filtrated and dried; a monodendron derivative witht-butoxycarbonyl group (Boc group)-protected amino groups was obtainedas a white solid (1.85 g yield 75%).

[0058] The identification results of the monodendron derivative areshown in Table 6. TABLE 6 ¹H—NMR spectra[CDCl₃, 500MHz, δ (ppm)]:1.40(s, 9H, —CH₃); 2.05(m, 2H, glu β-CH₂); 2.23(m, 2H, glu γ-CH₂):4.46(m, 1H, glu α-CH).

[0059] After dissolving the monodendron derivative (0.49 g, 2 mmol) andDCC (0.82 g, 4 mmol) in chloroform while stirring at 4° C. for 1 hour,the solution was added dropwise to a chloroform solution of compound [A](2.98 g, 5 mmol) and triethylamine (0.20 g, 2 mmol). After stirring thereaction mixture solution at 25° C. for 6 hours, the solution wasfiltrated using a glass filter (G4), the filtrate was concentrated underreduced pressure, and the product was purified by re-precipitation withmethanol. The precipitate was recovered by filtration, and was purifiedby silica gel column chromatography (solvent: chloroform/methanol -6/1(volume/volume)) to obtain the monodendron derivative (1.40 g, yield50%).

[0060] Production of the desired compound was confirmed by theappearance of an TR peak (1638 cm⁻¹) attributed to the amide bond.

[0061] The resulting monodendron derivative (1.40 g, 1 mmol) wasdissolved in trifluoroacetic acid (TFA), and the solution was stirredfor 1 hour to remove the protective group. The reaction solution wasrecrystallized from methanol at 4° C., and the crystal was filtrated anddried to obtain the monodendron derivative [J] (1.17 g, yield 90%).

[0062] Production of the desired compound was confirmed from thedisappearance of the methyl proton peaks (5-1.44) of the Boc group in¹H-NMR.

[0063] The monodendron derivative [J] (1.17 g, 0.9 mmol) was dissolvedin a mixed solution of chloroform and tetrahydrofuran (mixing ratio 1;1(volume/volume), and succinic anhydride (130 g, 1.35 mmol) was added,after which the solution was stirred for 5 hours. After removing thesolvent under a reduced pressure, the residue was recrystallized from amixed solution of ethanol and acetone (mixing ratio 1:5 (volume/volume))at 4° C. The crystal was filtrated and dried, whereby a carboxylic acidtetra-chain lipid [K] with a monodendron structure (0.95 g, yield 75%)was obtained.

INDUSTRIAL APPLICABILITY

[0064] As described in detail above, the present invention provides anon-phospholipid carboxylic acid-type lipid. This lipid is effective forcontrolling the surface charge density and surface polarity of thephospholipid vesicle and for preventing vesicle aggregating and fusingin the same manner as phospholipids, such as diacyiphosphatidyl glycerol(PG), diacylphosphatidyl inositol (PI), diacylphosphatidyl serine (PS)and diacylphosphatidyl ethanolamine (PE), which are generally used asmembrane lipids of conventional vesicles do, and may further be used asthe components of stable vesicle preparations for their excellentbiocompatibility. Further, when administered in the body, particularlyin the blood, this carboxyl acid-type lipid may also control theinteractions among plasma proteins and blood cell components.

[0065] Moreover, a carboxyl acid-type lipid that may easily besynthesized is provided, and since the structure of the hydrophobicmoiety can readily be changed, this carboxyl acid-type lipid may beapplied as negatively charged amphiphilic molecules suitable for the usein various applications such as emulsifiers, stabilizers, dispersingagents, solubilizing agents, mixing agents wetting agents, permeatingagents and viscosity modifiers in pharmaceuticals, food stuffs,cosmetics and dyes. Particularly, the lipid may be used as an artificialoxygen carrier if used as negatively charged lipids of vesiclescontaining hemoglobin.

1. A carboxylic acid-type lipid represented by the following generalformula (1]:

[wherein R₁, R₂ and R₃ represent substituents of which one isrepresented by the following general formula [x]:

(wherein M is a hydrogen atom or monovalent cation, and m is an integerof 1 to 5 that represents the methylene chain length), and the other twoare chained hydrocarbon groups; A₁, A₂ and A₃ are the same or differentsubstituents selected from the group consisting of C(O)O, CONH or NHCO;and n is an integer of 1 to 3 that represents the methylene chainlength).
 2. A carboxylic acid-type lipid represented by the followinggeneral formula (2):

[wherein R₁, R₂ and R₃ represent substituents of which one isrepresented by the following general formula [X]:

(wherein M is a hydrogen atom or monovalent cation, and m is an integerof 1 to 5 that represents the methylene chain length), and the other twoare chained hydrocarbon groups; A₁, A₂ and A₃ are the same or differentsubstituents selected from the group consisting of OC(O) and O; and nand n′ are integers of 1 to 3 that represent the methylene chainlength].
 3. A carboxylic acid-type lipid represented by the generalformula (3):

[wherein M is a hydrogen atom or monovalent cation; m is an integer of 1to 5 that represents the methylene chain length; R₁ and R₂ are chainedhydrocarbon groups; A₁ and A₂ are the same or different substituentsselected from the group consisting of C(O)O and CONH; and n is aninteger of 1 to 3 that represents the methylene chain length].
 4. Acarboxylic acid-type lipid represented by the following general formula[4]:

[wherein R₁, R₂ and R₃ represent substituents, of which one isrepresented by the following general formula [X]:

(wherein M is a hydrogen atom or monovalent cation, and m is an integerof 1 to 5 that represents the methylene chain length) and the other twoare chained hydrocarbon groups; A₁, A₂ and A₃ are the same or differentsubstituents selected from the group consisting of C(O)O and CONH; andR₄ is selected from the group consisting of a hydrogen atom, methylgroup and acetylene group].
 5. A carboxylic acid-type lipid representedby the following general formula [5]:

[wherein R₁, R₁ and R₃ are substituents of which one is represented bythe following general formula [X]:

(wherein M is a hydrogen atom or monovalent cation, and m is an integerof 1 to 5 that represents the methylene chain length), and the other twoare chained hydrocarbon groups; A₁, A₂ and A₃ are the same or differentsubstituents selected from the group consisting of OC(O), O, NH, CONHand NHCO; and D represents a saccharide].
 6. A carboxylic acid-typelipid represented by the following formula [6a] or [6b]

(wherein either one of R₁ or R₂ is a hydrogen atom and the other isOR′″; either one of R₃ and R₄ is a hydrogen atom and the other is OR″″;R, R′, R″, R′″, and R″″ are substituents of which one is represented bythe following general formula [Y];

(wherein M is a hydrogen atom or monovalent cation, A is CH₂ or CO, andm is an integer of 1 to 5 that represents the methylene chain length),at least two :are chained hydrocarbon groups, and the others arehydrogen atoms; and n is an integer of 1 to 3 that represents the degreeof polymerization].
 7. A carboxylic acid-type lipid represented by thefollowing general formula [7]:

(wherein R₁ is an aliphatic hydrocarbon group, F is a monodendronconstituting unit, R₂ is a linker, M is a hydrogen atom or monovalentcation, m is an integer of 1 to 5 that represents the methylene chainlength, p is an integer of 2, and q is an integer of 1 to 4 thatrepresents the number of repeating units in the dendron).
 8. Thecarboxylic acid-type lipid of claim 7, wherein the monodendronconstituting unit is one or more amino acid.
 9. A carboxylic acid-typelipid represented by the following general formula [8]:

(wherein M is a hydrogen atom or monovalent cation, m is an integer of 1to 5 that represents the methylone chain length, and n and n′ areintegers of 13 to 21 that represent the chain lengths of methylene).